Method and device for combined enrichment, processing and embedding of cytological specimens according to histological principles

ABSTRACT

A method and device for the processing of cytological specimens. The device comprises a preferably cone-shaped receptacle with a closed bottom part, sidewalls defining an upper aperture with a surface area larger than that of the bottom part, and a perforated membrane adapted to cover the aperture of the receptacle for retaining the cytological specimens. The membrane is preferably a mesh having a mesh size of up to 80 microns, the hydraulic conductance of the membrane being sufficient to allow the exchange of processing fluids when the receptacle is inserted into a tissue processing apparatus. The sidewalls form an obtuse angle with the bottom in order to promote such an exchange. In the method, the receptacle with specimens is inserted into a tissue processor with the membrane in a vertical position. After processing and paraffination, the receptacle is cooled allowing the specimens to sedimentate to form an enriched layer at the bottom of the receptacle. The solid paraffin block with enriched layer is thereafter removed from the receptacle and cut on a microtome.

BACKGROUND OF THE INVENTION

The present invention relates to the combined enrichment, processing andembedding of cytological specimens according to histological principles.

Most neoplastic and many other diseases are diagnosed by microscopicexamination of cytological cell or of histological tissue specimens. Newmethods for obtaining cytological specimens from tissues, e.g., byaspiration using fine needles and by brushing material off mucosalmembranes, have increased the demand for cytological methods that permitdiagnostic accuracy comparable to that of histological specimens.Cytological and histological preparations should be thin enough to letpass the incident light of the microscope after staining and they shouldpermit the use of all histochemical and immunological staining methodsthat are essential for accurate diagnoses.

Cytological specimens can be smeared onto slides and this originallyhaematological technique is used also for secretions and fine needleaspiration biopsies. Cells in fluids can be enriched onto filters (e.g.Millipore filters, Millipore Corporation, Bedford, Mass.) that permitthe fluid proper to pass; in this technique the number of applicablestaining methods is limited because the filters absorb many dyes. Cellscan be centrifuged onto glass slides. Disadvantages common to thesetechniques are that specimens may contain cell clusters so large thatthey cannot be analysed since enough light does not pass them afterstaining and, furthermore, clusters of cells cannot easily be dividedfor staining by special techniques.

The above draw-backs do not apply to histological techniques and someprefer to concentrate and collect valuable cytological specimens tobodies that can be processed and embedded histologically. Fluid gelatin,agar, gels and plasma have been used as "glues" that can be solidifiedto form bodies that can be processed histologically. Cell clusters havebeen collected into funnels prepared from filter paper or plastic(Nordgren Hans, not published) nets, or onto polycarbonate filters(Nordgren et al. APMIS 97:136-42, 1989) that can be processedhistologically. Fixed, repeatedly centrifuged and acetone-dehydratedspecimens have been paraffinized after evaporation of the acetone,concentration of the specimen being obtained by centrifugation in themelted paraffin, but the method is not suitable to mucinous specimens(Krogerus & Anderssson. Acta Cytol 32: 585-7, 1988) and seems laborious.

Histological tissue specimens, after fixation, are transferred through aseries of processing fluids that dehydrate and clear the tissues beforethey are impregnated by melted paraffin that upon cooling hardens to ablock that is cut by a microtome, the thin sections after staining beingexamined microscopically. The tissue pieces are generally transferred tosmall perforated and labelled tissue casettes, which during processingare kept in large perforated baskets. After paraffination, the tissuepieces are transferred manually to metal molds for embedding, the moldsbeing covered by the inverted labelled casette bottoms that afterhardening of the paraffin remain attached to the paraffin block, thecasette being clamped to the microtome jaws for cutting.

The latter laborious step of embedding has been considerably simplifiedby Pickett et al (U.S. Pat. No. 3,982,862). These authors transfer thefixed tissue piece directly into an open base pan, serving both as partof a tissue "casette" during paraffination and, after completedparaffination, as an embedding mold. For processing, the base pan isclosed by a perforated and labelled top member that, after processing ina manner similar to the casette bottoms above, remains attached to theparaffin block and fits the jaws of a microtome. Adequate paraffinationis achieved when, during processing, the top member closing the base panis positioned vertically. However, the method by Pickett et al. is asunsuitable for cytological specimens as are conventional histologicaltechniques.

In conventional automatic tissue processor types the baskets holding thecasettes are lifted into the air when transferred from one processingfluid to the next one. In some modern processor types the baskets arekept in one single closed compartment in which the fluids are exchanged,periods of low pressure being applied to speed up paraffination.

The technical disadvantages of cytological techniques were mentionedabove. From a cytological point of view the main disadvantages ofhistological techniques are that nucleated cells (diameter range some10-20 μm) and small cell clusters easily escape through histologicalcasette perforations (>1000 μm) and that specimens are not adequatelyenriched unless they are "glued" into pieces corresponding in size tohistological tissue ones.

SUMMARY OF THE INVENTION

The present invention describes a device and a method by which theaforementioned disadvantages may be eliminated and by which cytologicalspecimens, in a manner that involves no rough handling of fragilefragments, simultaneously are enriched and impregnated with paraffin toform paraffin blocks that can be sectioned also serially, permitting theuse of all the histochemical and immunological methods that often are ofkey importance for the diagnosis of human diseases.

The method according to the invention for processing cytologicalspecimens is characterized in that it comprises inserting the specimensto be processed into a cup-shaped receptacle having a closed bottompart, closed side-walls the upper edge of which define an upper aperturein the receptacle having an area larger than the area of the bottompart, the side-walls forming an obtuse angle with the said bottom part,the aperture is covered with a perforated membrane retaining thecytological specimens while still exhibiting a hydraulic conductancehigh enough as to allow the exchange of processing fluids therethrough,the receptacle with specimens is introduced in a tissue processingapparatus with the perforated membrane in the vertical position, afterprocessing the receptacle is turned so that the perforated membrane isin the horizontal position and directed upwards, thus permitting theenrichment of the specimens by sedimentation to form an enriched layerat the receptacle bottom, and after hardening, the paraffin block formedis removed from the receptacle for histological examination.

Though terms such as "paraffination" and "paraffin block" have and willbe used, the invention is not restricted to the use of paraffin waxproper but covers the use of also other substances with propertiessuitable for histological studies.

The perforated membrane covering said processing receptacle or cup andpreventing the specimen from escaping, in addition to being able towithstand moderate mechanical damage optimally should have perforationssmall enough to prevent small single nucleated cells (about 10 μm) fromescaping but a hydraulic conductance high enough to permit properexchange of processing fluids when other conditions, notably the shapeof the receptacle, favour such exchange. The fact that fluid flow ratherthan diffusion is responsible for exchange of processing fluids inautomatic tissue processors is indicated, e.g., by that fluid exchangethrough a membrane is very poor in a cup filled with fluid, closed by aperforated membrane and submerged vertically (membrane horizontal) withthe opening directed upwards in a larger vessel containing fluid withthe same or lower density, and far too poor to provide adequateparaffination.

The amount of fluid flow Q_(fluid) through a membrane with cylindricalperforations obeys Poiseuille's law, depending on the pressuredifference (P₁ -P₂) across the net and the hydraulic conductance perunit area of the membrane or ##EQU1## from which follows that flowthrough a perforated membrane can be increased by increasing the numberN of perforations per unit area, to the fourth power by increasing theperforation radius r, by decreasing the viscosity (visc.) of the fluidand by decreasing the length l of the perforations and by increasing thepressure difference. The radius of the perforations of commerciallyavailable histological perforated membranes (U.S. Pat. No. 3,982,862)and processing casettes (≧500 μm) is long compared to that of aperforated membrane, which holds back nucleated cells (radius about 5μm), but the latter's low hydraulic conductance due to the shortperforation radius must be compensated for by a vast number ofperforations per unit area, the use of a thin membrane and factors thatincrease the filtration pressure resulting in flow. The use of filtersemployed in cytology (e.g. Millipore Corporation, Bedford, Mass., U.S.A)was considered but these filters are too fragile for the purpose of theinvention. Nylon nets with mesh sizes ranging from 7 to 5000 μm arecommercially manufactured (Zuricher Beuteltuschfabrik Ag, Ruschlikon,Switzerland). Using the cups to be described below, poor paraffinationwas regularly obtained with the nets less than 10 μm (opening size 10×10μm), whereas adequate paraffination regularly was obtained with the 20μm net. Thus, depending on specimen type, a mesh size from 10 μm up, eg. up to 80 μm is contemplated in accordance with the invention, goodresults being obtained with nets 15 to 35 μm, a 20 μm net being optimalfrom several points of view. It is however conceivable to use the finertype nets if processing is enhanced, e.g. by employing vacuum in the newautomatic tissue processor types.

Under otherwise similar conditions, the hydraulic conductance of a"histological" perforation with a radius of 500 μm (e.g., U.S. Pat. No.3,982,862) is about 3.9×10⁶ higher than that of one with a radiuscalculated from the surface area (400 μm) of the mesh (20×20 μm) in theabove 20 μm net. This is, however, in part compensated for by otherproperties of the above nylon net, such as its open surface area of 16%,the about 40000 perforations/cm² (to be compared to U.S. Pat. No.3,982,862: open surface area 6.37% and 8.1 perforations/cm²) and afabric thickness of only 60 μm.

Though the properties of a net of the above described type made themethod of the invention possible, the invention is not restricted to theuse of that type of net or the indicated mesh-size and it covers alsothe use of fragile cytological filters supported by tougher wide-meshednets and other fabrics having properties corresponding to these nets.The perforated membrane will henceforth be termed net.

The invention also concerns a device or receptacle for the processing ofcytological specimens which comprises a cup with a closed bottom part,closed side-walls defining an upper aperture with a surface area largerthan that of the said bottom part, the side-walls forming an obtuseangle with the bottom part, as well as a perforated membrane adapted tocover the aperture of the cup for retaining the cytological specimens,the hydraulic conductance of the membrane being high enough as to allowthe exchange of processing fluids through the membrane when the cup isinserted in the tissue processing apparatus, the shape of the cuppromoting such exchange, and means for attaching the said membrane tothe cup.

The cup according to the invention can have a square (inverted frustrumof a pyramid) or rectangular cross-section but to facilitate the removalof the hardened paraffin, a cup with a frustoconical shape ispreferable, the net covering the said upper open larger base of thecone. An essential characteristic is, however, that the angle betweenthe closed bottom and the side-walls should be obtuse, and preferablywider than about 105° and preferably not larger than appr. 135°. A wideangle is essential because when the cup is covered with the said net andthe net is in the vertical position, fluid exchange is rather sluggishif the shape of the cup is cylindrical (angle between bottom andsidewalls 90°) and if it is deep. In conventional tissue processor typeswe have also experienced poor paraffination in cups in which the saidangle is 100° but never with angles >105°. Good paraffination isgenerally always obtained with angles between 105° and 120°. The widerthe angle the better is the fluid exchange. The fluid exchange is,according to tests performed, also driven by the hydrostatic pressurecreated by differences in fluid density on both sides of the perforatednet and by the movement of the baskets in conventional automatic tissueprocessors. In the latter, fluid does not flow out through the nets whennetted cups are lifted into the air while being transferred from oneprocessing fluid to the next one; fluid does, however, flow out of cupshalf-filled with fluid if the net above the fluid level is dry,indicating that the surface tension of the fluid in a wetted netprevents the hydrostatic pressure at the lower margin of the net fromleading to flow through the net. In those automatic tissue processors inwhich periods of vacuum are employed, fluid exchange is enhanced byvaporization of fluids; bubbles forming behind the net do not pass it,apparently due to the surface tension in the net, and the cups seem toempty; when the vacuum is relieved the cups refill with fluid. Thoughthis enhances fluid exchange, tests performed indicate that this doesnot suffice to ensure adequate paraffination if the cups are keptupright (nets horizontal) but when the nets are vertical adequateparaffination is obtained even with short (1 hour) paraffinationschedules.

The small bottom area of the cup also provides for the final enrichmentof the specimen in melted paraffin due to the fact that cells andfragments sedimentate from a comparatively large volume of fluid to acomparatively small bottom area. Tests have also shown that too wide anangle between the bottom and the sidewalls, i.e. above appr. 135°,prevents cells and fragments from gliding along the sidewalls to thebottom. Larger angles could be considered if the cup with specimens iscentrifuged.

The cup according to the invention serves both as a processingreceptacle and an embedding mold, in this respect resembling thebase-pan according to the U.S. Pat. No. 3,982,862, but in contrast tothe present invention, the specimen is not enriched to a small sectionarea in the known technique. Base pans with an 92°-100° angle betweenthe bottom and the side-walls have been proposed in that patent butsolely for the easy removal of the paraffin block. If used together withthe above 20 μm mesh size net those angles would probably result in poorparaffination of many specimens.

According to one preferred embodiment the said net can be clamped to theopen aperture of the said cup by a closing ring, secured to the cup bypincers, springs or similar devices which grip ridges or horizontal barson the cup.

Preferably the net can be incorporated to cover an opening in a separatebody or socket, the opening corresponding in size to the inner peripheryof the upper aperture of the cup and provided with a groove surroundingthe net that mates the outer periphery of the upper aperture of the cup,preventing the socket and the net from sliding away from the upperaperture of the cup.

According to a preferred embodiment, in both closing rings and thenetted bodies or sockets, henceforth termed net-pieces, the side-wallsof the opening extending above the nets form an angle with the surfaceof the net wider than the angle between the bottom of the cup and itsside-walls, in order to enhance that down- or uphill exchange ofprocessing fluids through the net that also determines the angle betweenthe bottom and the side-walls of the cups. The net-piece is preferablymade of material resistant to the processing fluids, having at least onesurface that can be labelled, thus forming a body that by a separatespring or pincers can be secured to ridges or bars on the cup. When theparaffin has hardened to a solid block, the net-piece with attachedparaffin block is removed from the cup, the outer dimensions of thenet-piece fitting the jaws of a microtome.

The cup according to the invention should preferably stand firmly whilethe specimen is introduced into it and during embedding. This can beachieved by using a cup-holder resting on a table surface and providedwith a hole corresponding in shape to the outer dimensions of the lowerpart of the cup. To enhance conductance of heat to the cup while thespecimen is enriched during embedding the cup-holder preferably is madeof metal. To prevent a conical cup from moving when the hardenedparaffin block with attached net-piece is removed with a twistingmovement from the cup, after gentle warming of the combined device inwarm tap water, the lower outer edge of the cup is preferably providedwith short horizontal bars that fit peripheral slits in the conical holeof the cup-holder.

The said springs or pincers used to secure the net-piece to the cup arepreferably provided with a device, preferably a hook that enables one toattach the combined net-piece with cup to the perforated side-walls ofthe baskets in automatic tissue processors so that the net remains inthe vertical position.

BRIEF DESCRIPTION OF THE DRAWING:

FIGS. 1A-1D demonstrates the principle of the cytological paraffinationand specimen enrichment method.

FIGS. 2A-2E shows details of the preferred cup and net-piece.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 shows the principle of the combined paraffination and enrichmentmethod for cytological specimens. The sedimented cytological specimen 1,preferably fixed or postfixed in formalin or some other preferablynon-coagulating fixative, is poured or transferred by a pipette into thecup 2, preferably having the shape of a frustrum of a cone the largebase of which forms an upper open aperture (FIG. 1A). The aperture isclosed by a fine-meshed net 3 which prevents the specimen from escapingbut has a hydraulic conductance high enough to permit exchange ofprocessing fluids when the net is kept in a vertical position, and theangle between the bottom of the cup and its side-walls is wide enough topromote a pressure on the net depending mainly on differences in densityof the fluids in- and outside the cup (FIG. 1B). The net is preferablyattached to a net-piece 4 in FIGS. 2 B-D that can be labelled. Afterprocessing the cup containing the specimen in melted paraffine is turnedin a vertical position onto a hot plate and the specimen enriches bysedimentation to form a layer covering the bottom of the cup (FIG. 1C),preferably after the net has been gently tapped upon, pierced using awarm scalpel and the specimen has been gently mixed to ensure its evendistribution (not shown in the Figure). Visible fragments sedimentate ina second or two, smaller ones according to Stokes' law more slowly butfrom a practical point of view 10 seconds have proved to ensure completesedimentation. Also centrifugation of the specimen in the meltedparaffin may be considered. After transfer of the cup to a cold platethe paraffin hardens and becomes fixed to the net-piece (not shown inFIG. 1) and the net-piece-paraffin cone is twisted off the cup, invertedand the specimen included in a top layer of the cone (FIG. 1D) is cut ona microtome.

FIG. 2 shows in natural size details of the preferred embodiment of thecup (A) and the net-piece (B-E) used for enrichment and paraffination ofcytological specimens. The cup, of which FIG. 2A shows a side-view, ispreferably made of a heat-conducting and non-corrosive metal. Thin wallswould facilitate conduction of heat but make it difficult to make theangle between the inner surface of the side-walls and the upper surfaceof the cup sharp enough to prevent from entering the space between thenet-piece and the cup. If plastic is used the bottom part of the cup canbe constructed so that it can be torn off, making the twisting-off ofthe paraffin cone unnecessary. The shape and dimensions of the cup canvary provided that the angle between the bottom and side-walls is wideenough (>105°) to promote exchange of the processing fluids. In thepreferred embodiment the cup has the shape of a frustrum of a cone, theradius of the upper open aperture being 9 mm and that of the bottom 4mm, the depth being 13 mm. These specific dimensions give an anglebetween the bottom and its side-walls of 111°, a compromise between thetheoretical arguments presented above. Cups with smaller and above alllarger bottom areas require consideration of the angle between thebottom and the side-walls. The upper part of the cup is provided with anencircling ledge 2', the upper part of which mates the correspondingindentation 5' in the net-piece 4, the lower edge fitting the arms of aspring that clamps the cup to the net-piece.

The net-piece 4 according to the invention (FIG. 2. B top view, Clongitudinal view, D bottom view, and E cross section with the cupproperly attached thereto) is preferably made of plastic and it isprovided with a central hole 5 covered by the net that preferably iswelded into or glued to it. The essentially rectangular net-piecepreferably has outer dimensions fitting the jaws of a microtome. Thenarrowest part of the central hole 5 is covered by the net 3 and has aradius equalling that of the upper aperture of the cup (2E). The anglebetween the net and the sidewalls of the cone-shaped hole 5" above thenet (FIGS. 2C and E), as well as the angle between the bottom of the cupand its sidewalls, has to be wide to enhance fluid exchange duringprocessing. After the net has been cut open at embedding and while theparaffin at the bottom of the cup is hardening the conical hole 5" isfilled with melted paraffin that helps to secure the paraffin cone tothe net-piece that will be clamped to the jaws of the microtome when thespecimen is sectioned. Beneath the net, the hole 5 the net-piece isprovided with an indentation 5' into which the ledge 2' of the cup fitsin a mating fashion. The net-piece can be clamped to the cup, preferablyusing a separate spring or pincers rather than hooks or other devices onthe net-piece that can be snapped to the cup. The net could also beattached to the cup by a closing ring.

As outlined above, the method is based on the fact that the net, thoughpreventing the specimen from escaping, has a hydraulic conductance highenough to make possible proper exchange of processing fluids. Such fluidexchange requires not only consideration of the hydraulic conductance ofthe net but also of the pressure that promote fluid exchange, a factorthat has influenced both the vertical position of the net duringprocessing and the angle between the bottom of the preferably conicalcup, and its sidewalls and the sidewalls of the upper aperture of thenet-piece.

The method described in the invention provides a number of advantages.Even small cell clusters in cytological specimens are enriched andparaffinized without being handled roughly and they can be cut on amicrotome and studied by all the methods that are available forhistological specimens embedded in paraffin. The specimens are enrichedonto so small a section area that no screening is required before afinal report is given by a pathologist. In the laboratory the method hasproved to be very valuable for fine needle aspiration, brush and similarspecimens.

What claimed is:
 1. A method for processing cytological specimenscomprising:inserting the specimens to be processed into a cup-shapedreceptacle having a closed bottom part and closed sidewalls, the closedsidewalls having an upper edge which defines an upper aperture in thereceptacle having an area larger than an area of the bottom part, thesidewalls forming an obtuse angle of about 105° to about 135° with thebottom part, the aperture being covered with a perforated membrane forretaining the cytological specimens, wherein the membrane is a meshhaving a mesh size up to 80 microns; inserting the receptacle withspecimens in a tissue processing apparatus containing processing fluidswith the perforated membrane in a vertical position so as to allow anexchange of processing fluids through the membrane and the introductionof paraffin; after processing and removal from the processing apparatus,the receptacle being turned so that the perforated membrane is in ahorizontal position and directed upwards, thus permitting the enrichmentof the specimens by sedimentation to form an enriched layer at thereceptacle bottom; and after allowing to harden to form a paraffin blockwith the enriched layer at the bottom, the block being removed from thereceptacle for histological examination.
 2. The method of claim 1,wherein the receptacle is of frustoconical shape.
 3. The method of claim2, wherein the angle between the sidewalls and the bottom part of thereceptacle is between about 105° and about 120°.
 4. The method of claim1, wherein the angle between the sidewalls and the bottom part of thereceptacle is between about 105° and about 120°.
 5. The method of anyone of claims 1, 2, 3 and 4, wherein the membrane has a mesh size ofbetween about 10 and about 80 μm.
 6. The method of claim 5, wherein themesh size is between about 15 and about 35 μm.
 7. The method of claim 6,wherein the mesh size is about 20 μm.
 8. The method of claim 1, whereinthe receptacle includes a means for attaching the membrane to thereceptacle, the attaching means comprising a socket positioned at theupper edge of the receptacle and having an opening which mates theaperture in the receptacle and the membrane is incorporated in theopening, and wherein the socket after processing and paraffination ofthe specimens and removal of the receptacle forms a support for thesolidified paraffin block when it is sectioned using a microtome.
 9. Themethod of claim 8, wherein the receptacle has ridges on its outersidewalls and the socket is attached to the receptacle by means forengaging the ridges.
 10. The method of claim 8, wherein the opening ofthe socket has sidewalls above the membrane which form an angle with themembrane which is bigger than the angle between the sidewalls and thebottom part of the receptacle.
 11. The method of claim 1, wherein thereceptacle is made of plastic and has a removable plastic bottom part.